When conducting genetic manipulation or other reactive engagement with or treatment of cells or tissue, it may be necessary to partially or completely penetrate the cell walls and/or membranes with a biological or other agent in order to achieve a desired affect on the cell wall and/or internal cellular elements such as cytoplasm, nuclei, plastids, chromosomes, plasmids, etc. to achieve a desired objective. Such objectives may include, for example, the destruction of selected elements, the production of new or improved biological performance characteristics, the modification of a particular microbe or plant variety to control color, growth rate, disease resistance or protein production, the tagging of cells for tracking and identification or the micromanipulation of cell by in situ rotation or displacement in space. Often such penetration is accomplished by applying the biological or other agent to carrier particles that are impressed on the cells.
In genetic research, for example, such methods are used to penetrate tissue and cells with particles precoated with plasmid DNA encoding genes of interest; cell penetration is followed by DNA delivery into the cell nucleus. To reach the intracellular space and then the cell nucleus, the particles have to traverse formidable cell walls and membranes. Because these cell walls are so hard to penetrate, the particles carrying the DNA are actually driven into the cells by the force of an explosive or an electrical discharge so that the kinetically driven particles smash into the target tissue. Even then, in order to have the necessary energy for penetration, the particles have to be several micrometers in diameter. Thus, the implantation process results in appreciable cell damage due to the impact of the particles and/or due to sonic concussion from the particle-propelling discharge. Some cell tissue, drawing upon its natural strength, may recover from this trauma sufficiently to integrate the newly delivered genetic material into its chromosomes in the nuclei; however a large percentage of the tissue is not able to do so.
These prior methods of delivering such particles also lack sufficient control over particle size distribution, particle coating quality and the velocity and direction of travel of the particles, resulting in lack of predictability and reproducibility of the particle implantation. The prior delivery techniques are further disadvantaged because they require that the target tissue be maintained in a vacuum which may remove moisture from the treated tissue contributing to tissue degradation. Moreover, the apparatus for performing the implantations are time-consuming to set up prior to each implantation cycle and difficult to clean after same so that the throughputs of the apparatus are relatively low.
Other methods employed or suggested for direct gene delivery to cells include the use of microlasers, microbead vortexing, electrofusion, chemical fusion, microinjection and electropotation. Such techniques all rely on increasing the permeability of the tissue cells by physically, chemically or electrically disrupting cell walls and/or membranes temporarily; exogeneously added DNA may then enter the cell through the temporary ruptures. Some of these methods, including microinjection and fusion of preselected protoplasts or subprotoplasts require working at the single cell level. This necessitates micromanipulation of the cells, often involving immobilization by agarose plating or pipette suction. Such micromanipulations must be carried out with a microscope placed in the sterile environment of a laminar flow hood, which can be very cumbersome. Also, controlled fusion, for example in the production of somatic hybrids, requires bringing the fusion partners into close proximity which, to this day, is still technically difficult to accomplish.